Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness.
In the present study we tested the responsiveness of human corneal epithelial cells (HCECs) and corneal fibroblasts to lipopolysaccharide (LPS), a Toll-like receptor (TLR) 4 ligand. Purified Pseudomonas aeruginosa LPS was used to stimulate telomerase-immortalized HCECs (HUCL) and stromal fibroblast (THK) cell lines. Exposure of cells to LPS induced a time-dependent activation of NF-kappaB in THK but not in HUCL cells, as assessed by an increase in IkappaB-alpha phosphorylation and degradation. Concomitant with NF-kappaB activation, LPS-treated THK cells, but not HUCL cells, produced a significantly larger number of cytokines than control untreated cells. A cell surface biotinylation assay revealed that HUCL cells express TLR4 intracellularly, whereas TLR5 is expressed on the cell surface. Furthermore, reverse transcriptase-PCR analysis revealed that HUCL and primary HCECs, in contrast to THK cells, do not express myeloid differentiation (MD)-2. Thus, our results demonstrate that the LPS unresponsiveness of HCECs might be due to deficient expression of MD-2, an essential component for LPS-TLR4 signaling.
Zhang
Jing
J
Department of Ophthalmology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, MI 48201, USA.
Kumar
Ashok
A
Wheater
Michelle
M
Yu
Fu-Shin X
FS
D015153:Blotting, Western; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015536:Down-Regulation / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96